Western blot analysis of BDNF expression in human SH-SY5Y cell lysates (A) and human brain (B). Polyclonal rhBDNF antibody R-1707-100 (1 µg/mL) detects monomeric BDNF at 14 kDa, and monomeric proBDNF at 32 kDa in lysates prepared either in RIPA buffer or in acid-extraction buffer (A). Control antibody M-1744-50/100 (1 µg/mL) confirms detection of mature BDNF (Lane 1) and proBDNF (Lane 2) using BDNF and proBDNF proteins. A second band at 18 kDa is observed in the proBDNF standard, likely to represent a proBDNF degradation product. In human brain (B), R-1707-100 detects mature BDNF (14 kDa) and proBDNF (32 kDa) in both preparations (Tris-buffer and acid-treated). Acid-treated brain demonstrates lower background staining and gives a stronger BDNF signal.
Western Blotting Method: SDS-PAGE: denaturing and reducing, 12% Bis-Tris gel; Transfer: Tris-Glycine buffer, semi-dry transfer; Membrane: nitrocellulose (0.22 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibody: overnight at 4°C; Secondary antibody: anti-mouse-HRP or anti-rabbit-HRP (1/6000), 2 hours at RT; Detection: Chemiluminiscence.